Acetylcholinesterase (ACHE) and butyrylcholinesterase (BuChE) are hydrolyzing enzymes present in various human or animal tissues, including plasma, muscles and brain. AChE functions primarily to hydrolyze acetylcholine and is essential to proper neuronal and neuromuscular activity (e.g., in regulation of chemical synapses between neurons and in neuromuscular junctions). BuChE is a serum cholinesterase with a broad hydrolytic spectrum that provides protection against a variety of AChE inhibitors. A similar end may be achieved by a variant of AChE found on the membranes of erythrocytes. Both enzymes are believed to serve as circulating scavengers for anti-AChE agents in protection of the vital synaptic ACHE. Therefore, administration of cholinesterases has the ability to boost natural human ability to counteract the toxic effects of anti-cholinergic agents.
While AChE and BuChE are both cholinesterases that may be used to counteract the toxic effects of anti-cholinergics and other toxic agents, their biochemical properties are distinct. Further, the amino acid sequences of the two enzymes are only 50% identical, with critical differences in several key positions.
For example, AChE displays nearly 100-fold selectivity toward acetylcholine over the longer chain butyrylcholine. Most of this increase is due to a 50-fold increase of Kcat (a measure of catalytic efficiency) and only 2-fold increase in the Km (a measure of substrate affinity). Conversely, BuChE has no significant substrate selectivity with both Km and Kcat nearly the same for both substrates. In addition AChE is inhibited by substrate inhibition above 2 mM, while BuChE is activated by substrate concentrations in the range of 20-40 mM. In addition, BuChE is reactive against a variety of substrates, for example, cocaine, for which ACHE is practically refractory.
Various compounds are well known to inhibit the hydrolyzing activity of human cholinesterases. Exposure to such anti-cholinesterase agents leads to over-stimulation of cholinergic pathways, causing muscular tetany, autonomous dysfunction and, potentially, death. While some naturally-occurring cholinesterase inhibitors are very potent, human exposure to them is rare. However, man-made anti-cholinesterase compounds, especially organophosphates (OPs), are widely used as pesticides and pose a substantial occupational and environmental risk. Even more ominous is the fear of deliberate use of OPs as chemical warfare agents against individuals or populations.
Availability of an agent specific cholinesterase provides a more effective treatment of anti-cholinergic response because AChE and BuChE differ in their sensitivity to many inhibitors. For example, BuChE is much more sensitive to the organophosphate tetraisopropyl pyrophosphoramide (Iso-OMPA), while ACHE is generally much more sensitive to cholinesterase inhibitors such as Diisopropylfluoro-phosphate (DFP) and 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, (BW284c51). The Ki, (or measure of inhibitor efficiency) against BW284c51 for ACHE is 10 nM (nano moles/liter), and for BuChE is 14,000 nM (or 14 μM), which represents a 1400-fold difference in sensitivity of BuChE compared to AChE.
Current medical interventions, in the case of acute exposure to anti-cholinesterase agents, include use of the muscarinic receptor antagonist, atropine, and oximes to reactivate the OP-modified cholinesterase. The reversible carbamate, pyridostigmine bromide, is also used as a prophylactic. However, these conventional treatments have limited effectiveness and have serious short- and long-term side effects. In fact, the routine treatments, while successfully decreasing anti-cholinesterase-induced lethality, rarely alleviate post-exposure delayed toxicity, which may result in significant performance deficits and even permanent brain damage.
A different approach in treatment and prevention of anti-cholinesterase toxicity seeks to mimic one of the physiological lines of defense against such agents present in mammals. The efficacy of this treatment to protect against a challenge of OPs was tested in a variety of animal models, such as mice, rats, guinea pigs, and primates, and was found to be comparable to or better than the currently-used drug regimens in preventing OP-induced mortality without any detrimental side-effects.
Naturally-occurring cholinesterases in human plasma are known to be important in metabolizing systemic toxins and have been tested in a range of animal models, particularly in cocaine detoxification. Naturally-occurring levels in the human body are limited in therapeutic applications, because these levels are so low. Genetic modification of natural cholinesterases to improve catalytic efficiency has shown promise as treatment for drug detoxification. More specifically, recombinant BuChE, produced using bacterial transformation, and then transfected into human kidney cells, was shown to increase cocaine hydrolysis. Though cholinesterases are known to be effective as anti-neurotoxins, the largest limitation in use of ChEs is the cost-effective production of sufficient quantities.
Despite the promise of cholinesterases as an effective treatment against nerve-agent intoxication and other toxins, the practicality of this therapeutic approach depends on the availability of large amounts of these enzymes, which are required in stoichiometric rather than catalytic quantities. Currently, human-plasma derived BuChE has been identified by the US military as a first generation candidate to go into human clinical trials. However, a reliable, safe, non-supply-limited and inexpensive source of ChEs is still needed, because a stock pile of 1 kg of pure enzyme would require dedicating the whole annual US supply of outdated plasma to a purification effort at an enormous cost.
Genetically-engineered plants have recently been recognized as one of the most cost-effective means for the production of useful recombinant proteins and pharmaceuticals. Expressing human enzymes, and more particularly human acetylcholinesterase, in plants is known in the art; however, no system or method has yet been disclosed for optimizing human BuChE-enzyme expression in plants. Therefore, a need exists for a method of optimizing human gene expression of human BuChE in plants and, more specifically, for a method for increasing the levels of expression of human BuChE enzymes in plants by optimizing the expression constructs that encode the expression constructs for expression in plants.